KR20080044277A - Combination of organic compounds - Google Patents

Abstract

The present invention

a) pyrimidylaminobenzamide compound; And

b) HDAC inhibitor compounds

Pharmaceutical combinations, and methods of treating or preventing proliferative diseases using such combinations.

Description

Combination of Organic Compounds {COMBINATION OF ORGANIC COMPOUNDS}

The present invention provides a pharmaceutical combination comprising a pyrimidylaminobenzamide compound and a histone deacetylase inhibitor and the above in proliferative diseases such as, for example, tumors, myeloma, leukemia, psoriasis, restenosis, percutaneous dermatitis and fibrosis. It relates to the use of the combination.

Despite the numerous treatment options for patients with proliferative diseases, there remains a need for effective and safe antiproliferative agents and their preferential use in combination therapy.

Reversible acetylation of histones is a major regulator of gene expression that acts by altering transcription factor access to DNA. In normal cells, histone deacetylase (HDAC) and histone acetyltransferase together control the acetylation levels of histones to be balanced. Inhibition of HDAC accumulates hyperacetylated histones, resulting in various cellular responses. Inhibitors of HDAC have been studied for therapeutic effects on cancer cells. Recent advances in the field of HDAC inhibitor research have provided very active and stable active compounds suitable for treating tumors.

Summary of the Invention

In accordance with the present invention, a combination comprising at least one pyrimidylaminobenzamide compound and an HDAC inhibitor (eg, as defined below) can be applied to tumors, myeloma, leukemia, psoriasis, restenosis, percutaneous dermatitis and fibrosis It has been found that it has a beneficial effect on proliferative diseases such as.

<Pyrimidylaminobenzamide compound>

The present invention relates to the use of pyrimidylaminobenzamide compounds of formula (I) and N-oxides or pharmaceutically acceptable salts thereof for the preparation of pharmaceutical compositions for the treatment of kinase dependent diseases.

Where

R ₁ represents hydrogen, lower alkyl, lower alkoxy-lower alkyl, acyloxy-lower alkyl, carboxy-lower alkyl, lower alkoxycarbonyl-lower alkyl or phenyl-lower alkyl;

R ₂ is hydrogen, lower alkyl, cycloalkyl, benzcycloalkyl, heterocyclyl, aryl group optionally substituted by one or more identical or different radicals R ₃ , or 0, 1, 2 or 3 ring nitrogen atoms, 0 Or a mono- or bicyclic heteroaryl group comprising one oxygen atom and zero or one sulfur atom, in which case the group is mono- or polysubstituted or unsubstituted; R ₃ is hydroxy, lower alkoxy, acyloxy, carboxy, lower alkoxycarbonyl, carbamoyl, N-monosubstituted or N, N-disubstituted carbamoyl, amino, mono- or di-substituted amino, cycloalkyl, Heterocyclyl, aryl groups, or mono- or bicyclic heteroaryl groups containing 0, 1, 2 or 3 ring nitrogen atoms, 0 or 1 oxygen atoms and 0 or 1 sulfur atoms, or in each case The group is mono- or polysubstituted or unsubstituted); or

R ₁ and R ₂ together are optionally worked by lower alkyl, cycloalkyl, heterocyclyl, phenyl, hydroxy, lower alkoxy, amino, mono- or disubstituted amino, oxo, pyridyl, pyrazinyl or pyrimidinyl Substituted or disubstituted alkylene having 4, 5 or 6 carbon atoms; Benzalkylene having 4 or 5 carbon atoms; Oxaalkylene with one oxygen and three or four carbon atoms; Or azaalkylene having 1 nitrogen and 3 or 4 carbon atoms, wherein nitrogen is lower alkyl, phenyl-lower alkyl, lower alkoxycarbonyl-lower alkyl, carboxy-lower alkyl, carbamoyl-lower alkyl, N Unsubstituted or substituted with mono- or N, N-disubstituted carbamoyl-lower alkyl, cycloalkyl, lower alkoxycarbonyl, carboxy, phenyl, substituted phenyl, pyridinyl, pyrimidinyl, or pyrazinyl) Represents;

R ₄ represents hydrogen, lower alkyl, or halogen.

The general terms used above and below preferably have the following meanings in the context of the present application, unless otherwise indicated.

The prefix "lower" indicates that the radical, linear or branched with single or multiple branches, has up to seven, in particular up to four carbon atoms.

Where plural forms are used for compounds, salts, etc., this should also be taken to mean a single compound, salt, or the like.

Any asymmetric carbon atom may be present in the (R)-, (S)-or (R, S) -configuration, preferably in the (R)-or (S) -configuration. Thus, the compounds may exist as mixtures of isomers or as pure isomers, preferably enantiomer-pure diastereomers.

The invention also relates to possible tautomers of the compounds of formula (I).

Lower alkyl is alkyl having 1 to 7, preferably 1 to 4 carbons, linear or branched; Preferably, lower alkyl is butyl (eg n-butyl, sec-butz, isobutyl, tert-butyl), propyl (eg n-propyl or isopropyl), ethyl or methyl. Preferably lower alkyl is methyl, propyl or tert-butyl.

Lower acyl is preferably formyl or lower alkylcarbonyl, in particular acetyl.

An aryl group is an aromatic radical bonded to a molecule via a bond located at an aromatic ring carbon atom of the radical. In a preferred embodiment, aryl is an aromatic radical having 6 to 14 carbon atoms, in particular phenyl, naphthyl, tetrahydronaphthyl, fluorenyl or phenanthrenyl, in particular amino, mono- or di-substituted amino Halogen, lower alkyl, substituted lower alkyl, lower alkenyl, lower alkynyl, phenyl, hydroxy, etherified or esterified hydroxy, nitro, cyano, carboxy, esterified carboxy, alkanoyl, benzoyl, Carbamoyl, N-monosubstituted or N, N-disubstituted carbamoyl, amidino, guanidino, ureido, mercapto, sulfo, lower alkylthio, phenylthio, phenyl-lower alkylthio, lower alkylphenyl Thio, lower alkylsulfinyl, phenylsulfinyl, phenyl-lower alkylsulfinyl, lower alkylphenylsulfinyl, lower alkylsulfonyl, phenylsulfonyl, phenyl-lower alkylsulfonyl, lower alkylphenylsulfonyl, halogen-lower al Kilmercapto, Hal Gen-lower alkylsulfonyl (e.g., methanesulfonyl, especially trifluoromethyl), di-hydroxy-violet (-B (OH) _2), heterocyclyl, a mono- adjacent the C- or bicyclic heteroaryl group, and the ring Unsubstituted or substituted by one or more, preferably up to three, in particular one or two substituents selected from lower alkylene dioxys (eg methylene dioxy) bonded to the atom. Aryl is more preferably phenyl, naphthyl or tetrahydronaphthyl, which in each case is halogen (in particular fluorine, chlorine or bromine); Hydroxy; Hydroxy etherified by lower alkyl (eg methyl), halogen-lower alkyl (eg trifluoromethyl), or phenyl; Lower alkylene dioxy (eg methylenedioxy), lower alkyl (eg methyl or propyl) bonded to two adjacent C-atoms; Halogen-lower alkyl (eg trifluoromethyl); Hydroxy-lower alkyl (eg hydroxymethyl or 2-hydroxy-2-propyl); Lower alkoxy-lower alkyl (eg methoxymethyl or 2-methoxyethyl); Lower alkoxy carbonyl-lower alkyl (eg, methoxycarbonylmethyl); Lower alkynyl (eg 1-propynyl); Esterified carboxy, in particular lower alkoxycarbonyl (eg methoxycarbonyl, n-propoxy carbonyl or iso-propoxy carbonyl); N-monosubstituted carbamoyl, in particular carbamoyl monosubstituted by lower alkyl (eg methyl, n-propyl or iso-propyl); Amino; Lower alkylamino (eg, methylamino); Di-lower alkylamino (eg, dimethylamino or diethylamino); Lower alkylene-amino (eg, pyrrolidino or piperidino); Lower oxaalkylene-amino (eg morpholino), lower azaalkylene-amino (eg piperazino), acylamino (eg acetylamino or benzoylamino); Lower alkylsulfonyl (eg, methylsulfonyl); Sulfamoyl; Or independently substituted or unsubstituted by one or two substituents selected from the group comprising phenylsulfonyl.

The cycloalkyl group is preferably cyclopropyl, cyclopentyl, cyclohexyl or cycloheptyl and is unsubstituted or at least one, in particular one or two substituents, most preferably selected from the group defined above as substituents for aryl Is substituted by lower alkyl (e.g. methyl), lower alkoxy (e.g. methoxy or ethoxy), or hydroxy, and further by oxo, or in the benzo ring, e.g., in benzcyclopentyl or benzcyclohexyl Can be fused.

Substituted alkyl is alkyl as defined last, in particular lower alkyl, preferably methyl; Mainly halogen (in particular fluorine), amino, N-lower alkylamino, N, N-di-lower alkylamino, N-lower alkanoylamino, hydroxy, cyano, carboxy, lower alkoxycarbonyl and phenyl- There may be at least one, in particular up to three substituents selected from lower alkoxycarbonyls. Trifluoromethyl is particularly preferred.

Mono- or di-substituted aminos are, in particular, lower alkyl (eg, methyl); Hydroxy-lower alkyl (eg, 2-hydroxyethyl); Lower alkoxy lower alkyl (eg methoxy ethyl); Phenyl-lower alkyl (eg benzyl or 2-phenylethyl); Lower alkanoyl (eg acetyl); Benzoyl; Substituted benzoyl, wherein the phenyl radical is in particular nitro, amino, halogen, N-lower alkylamino, N, N-di-lower alkylamino, hydroxy, cyano, carboxy, lower alkoxycarbonyl, lower alkanoyl and carbo Substituted by one or more, preferably one or two substituents selected from bamoyl); And phenyl-lower alkoxycarbonyl, wherein the phenyl radical is in particular nitro, amino, halogen, N-lower alkylamino, N, N-di-lower alkylamino, hydroxy, cyano, carboxy, lower alkoxycarbonyl, lower Amino substituted by one or two radicals independently selected from each other from one or more, preferably one or two substituents selected from alkanoyl and carbamoyl; Preferably N-lower alkylamino (eg N-methylamino), hydroxy-lower alkylamino (eg 2-hydroxyethylamino or 2-hydroxypropyl), lower alkoxy lower alkyl (eg methoxy ethyl ), Phenyl-lower alkylamino (e.g. benzylamino), N, N-di-lower alkylamino, N-phenyl-lower alkyl-N-lower alkylamino, N, N-di-lower alkylphenylamino, lower alka Noylamino (eg, acetylamino) or a substituent selected from the group comprising benzoylamino and phenyl-lower alkoxycarbonylamino, wherein the phenyl radical is in each case particularly with nitro or amino, and also halogen, amino, Unsubstituted or substituted by N-lower alkylamino, N, N-di-lower alkylamino, hydroxy, cyano, carboxy, lower alkoxycarbonyl, lower alkanoyl, carbamoyl or aminocarbonylamino Neunda. Disubstituted aminos can also be lower alkylene-amino (eg, pyrrolidino, 2-oxopyrrolidino or piperidino); Lower oxaalkylene-amino (eg morpholino), or lower azaalkylene-amino (eg piperazino) or N-substituted piperazino (eg, N-methylpiperazino or N-methoxycarbonylpiperazino).

Halogen is especially fluorine, chlorine, bromine or iodine, especially fluorine, chlorine or bromine.

The etherified hydroxy is in particular C ₈ -C ₂₀ alkyloxy (eg n-decyloxy), lower alkoxy (preferably) (eg methoxy, ethoxy, isopropyloxy or tert-butyloxy), phenyl Lower alkoxy (eg benzyloxy, phenyloxy), halogen-lower alkoxy (eg trifluoromethoxy, 2,2,2-trifluoroethoxy or 1,1,2,2-tetrafluoroethoxy ), Or lower alkoxy, preferably imidazolyl (eg 1H-imidazol-1-yl), pyrrolyl, benz substituted by mono- or bicyclic heteroaryl containing one or two nitrogen atoms Imidazolyl (eg 1-benzimidazolyl), pyridyl (particularly 2-, 3- or 4-pyridyl), pyrimidinyl (particularly 2-pyrimidinyl), pyrazinyl, isoquinoli Lower alkoxy substituted by nil (especially 3-isoquinolinyl), quinolinyl, indolyl or thiazolyl.

The esterified hydroxy is in particular lower alkanoyloxy, benzoyloxy, lower alkoxy carbonyloxy (eg tert-butoxycarbonyloxy), or phenyl-lower alkoxycarbonyloxy (eg benzyloxycarbonyloxy) .

The esterified carboxy is in particular lower alkoxycarbonyl (eg tert-butoxycarbonyl, iso-propoxycarbonyl, methoxycarbonyl or ethoxycarbonyl), phenyl-lower alkoxycarbonyl, or phenyloxycarbonyl to be.

Alkanoyl is mainly alkylcarbonyl, in particular lower alkanoyl (eg acetyl).

N-monosubstituted or N, N-disubstituted carbamoyl is in particular lower alkyl, phenyl-lower alkyl and hydroxy-lower alkyl, or lower alkylene, oxa-lower alkylene or aza- optionally substituted at terminal nitrogen atoms. Substituted by one or two substituents independently selected from lower alkylene.

Mono- or bicyclic heteroaryl groups comprising 0, 1, 2 or 3 ring nitrogen atoms, 0 or 1 oxygen atoms and 0 or 1 sulfur atoms (in each case said groups are unsubstituted, mono- or multi-substituted) Substituted) represents an unsaturated heterocyclic moiety in the ring where the heteroaryl radical is attached to the rest of the molecule of formula (I), preferably at least one carbon atom in the bonding ring as well as optionally in any annealed ring is nitrogen, oxygen And a ring substituted by a heteroatom selected from the group consisting of sulfur; Wherein the bonding ring preferably has 5 to 12, more preferably 5 or 6 ring atoms; It is unsubstituted or by one or more, in particular one or two substituents selected from the group defined above as substituents for aryl, most preferably lower alkyl (eg methyl), lower alkoxy (eg methoxy Or ethoxy), or hydroxy. Preferably mono- or bicyclic heteroaryl groups are 2H-pyrrolyl, pyrrolyl, imidazolyl, benzimidazolyl, pyrazolyl, indazolyl, purinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl , 4H-quinolinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalyl, quinazolinyl, quinolinyl, pterridinyl, indolinyl, 3H-indolyl, indolyl, iso Indolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, furazanyl, benzo [d] pyrazolyl, thienyl and furanyl. More preferably, the mono- or bicyclic heteroaryl group is pyrrolyl, imidazolyl (eg 1H-imidazol-1-yl), benzimidazolyl (eg 1-benzimidazolyl), indazolyl ( In particular 5-indazolyl), pyridyl (particularly 2-, 3- or 4-pyridyl), pyrimidinyl (particularly 2-pyrimidinyl), pyrazinyl, isoquinolinyl (particularly 3- Isoquinolinyl), quinolinyl (particularly 4- or 8-quinolinyl), indolyl (particularly 3-indolyl), thiazolyl, benzo [d] pyrazolyl, thienyl and furanyl Selected from the group. In one preferred embodiment of the invention, the pyridyl radical is substituted by hydroxy at the ortho position relative to the nitrogen atom and thus is at least partially present in the form of the corresponding tautomers which are pyridin- (1H) 2-one. In another preferred embodiment, the pyrimidinyl radical is substituted by hydroxy in both 2 and 4 positions and thus exists in various tautomeric forms, for example pyrimidine- (1H, 3H) 2,4-dione. .

Heterocyclyl is a 5, 6 or 7 membered heterocyclic system having one or two heteroatoms, in particular selected from the group comprising nitrogen, oxygen and sulfur, which may be unsaturated, fully or partially saturated, and substituted Or is especially substituted by lower alkyl (eg methyl), phenyl-lower alkyl (eg benzyl), oxo, or heteroaryl (eg 2-piperazinyl); Heterocyclyl is especially 2- or 3-pyrrolidinyl, 2-oxo-5-pyrrolidinyl, piperidinyl, N-benzyl-4-piperidinyl, N-lower alkyl-4-piperidinyl, N Lower alkyl-piperazinyl, morpholinyl (eg 2- or 3-morpholinyl), 2-oxo-1H-azin-3-yl, 2-tetrahydrofuranyl or 2-methyl- 1,3-dioxolan-2-yl.

Salts are in particular pharmaceutically acceptable salts of compounds of formula (I).

Such salts are formed, for example, from compound of formula (I) having a basic nitrogen atom, as acid addition salts with organic or inorganic acids, in particular pharmaceutically acceptable salts. Suitable inorganic acids are, for example, halogen acids such as hydrochloric acid, sulfuric acid or phosphoric acid. Suitable organic acids are, for example, carboxylic acids, phosphonic acids, sulfonic acids or sulfamic acids, for example acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid, succinic acid, adipic acid, pimelic acid , Suveric acid, azelaic acid, malic acid, tartaric acid, citric acid, amino acids such as glutamic acid or aspartic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, cyclohexanecarboxylic acid, adamantanecarboxylic acid, benzoic acid, salicylic acid , 4-aminosalicylic acid, phthalic acid, phenylacetic acid, mandelic acid, cinnamic acid, methane- or ethane-sulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 1, 5-naphthalene-disulfonic acid, 2-, 3- or 4-methylbenzenesulfonic acid, methyl sulfuric acid, ethyl sulfuric acid, dodecyl sulfuric acid, N-cyclohexyl sulfamic acid, N-methyl-, N-ethyl- or N-propyl- Sulfamic acid, or other organic protic acid, such as ascorbic acid.

In the presence of negatively charged radicals such as carboxy or sulfo, salts can also be formed by reaction with bases, for example metal or ammonium salts such as alkali metal salts or alkaline earth metal salts such as sodium, potassium, magnesium Or calcium salts or ammonium salts with ammonia or a suitable organic amine such as tertiary monoamines such as triethylamine or tri (2-hydroxyethyl) amine, or heterocyclic bases such as N-ethyl Salts with -piperidine or N, N'-dimethylpiperazine.

When basic and acidic groups are present in the same molecule, the compounds of formula (I) can also form internal salts.

It is also possible to use pharmaceutically unacceptable salts, for example picrates or perchlorates, for isolation or purification purposes. For therapeutic use, only pharmaceutically acceptable salts or free compounds are used (if applicable in the form of pharmaceutical preparations) and therefore they are preferred.

In view of the close relationship between the novel compounds in their free form and the novel compounds in their salt form, including salts that can be used as intermediates, any reference to the above and below free compounds, for example, in the purification or identification of new compounds Is understood to refer to the corresponding salt as appropriate for convenience.

Compounds in the range of formula (I) and methods for their preparation are disclosed in WO 04/005281, published January 15, 2004, which is incorporated herein. Preferred compounds are 4-methyl-3-[[4- (3-pyridinyl) -2-pyrimidinyl] amino] -N- [5- (4-methyl-1H-imidazole having the structure of formula II: -1-yl) -3- (trifluoromethyl) phenyl] benzamide (hereinafter referred to as "Compound (II)").

<HADC inhibitor compound>

Of particular interest for use in the combinations of the present invention are HDAC compounds, which are the hydroxyxamate compounds represented by the general formula (III) below or pharmaceutically acceptable salts thereof.

Where

R ₁ is H, halo or straight-chain C ₁ -C ₆ alkyl (in particular methyl, ethyl or n-propyl, wherein the methyl, ethyl and n-propyl substituents are substituted by one or more substituents described below for alkyl substituents Unsubstituted);

R ₂ is H, C ₁ -C ₁₀ alkyl, preferably C ₁ -C ₆ alkyl (eg methyl, ethyl or —CH ₂ CH ₂ —OH), C ₄ -C ₉ cycloalkyl, C _4- C ₉ heterocycloalkyl, C ₄ -C ₉ heterocycloalkylalkyl, cycloalkylalkyl (eg cyclopropylmethyl), aryl, heteroaryl, arylalkyl (eg benzyl), heteroarylalkyl (eg For example, pyridylmethyl),-(CH ₂ ) _n C (O) R ₆ ,-(CH ₂ ) _n OC (O) R ₆ , amino acyl, HON-C (O) -CH = C (R ₁ ) -Aryl-alkyl- and-(CH ₂ ) _n R ₇ ;

R ₃ and R ₄ are the same or different and are independently H, C ₁ -C ₆ alkyl, acyl or acylamino, or

R ₃ and R ₄ together with the carbon to which they are attached represent C═O, C═S or C═NR ₈ ; or

R ₂ together with the nitrogen to which it is attached, and R ₃ together with the carbon to which it is attached, C ₄ -C ₉ heterocycloalkyl, heteroaryl, polyheteroaryl, non-aromatic polyheterocycle or aryl and non-aryl polyhetero Can form a cycle of mixed rings;

R ₅ is H, C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, acyl, aryl, heteroaryl, arylalkyl (eg, benzyl), heteroarylalkyl ( For example, pyridylmethyl), aromatic polycycles, non-aromatic polycycles, mixed aryl and non-aryl polycycles, polyheteroaryls, non-aromatic polyheterocycles, and mixed aryl and non-aryl polyheteros Selected from cycles;

n, n ₁ , n ₂ and n ₃ are the same or different and are independently selected from 0 to 6, and when n ₁ is 1 to 6 each carbon atom is independently selected from R ₃ and / or R ₄ Can be substituted;

X and Y are the same or different and H, halo, C ₁ -C ₄ alkyl, such as CH ₃ and CF ₃ , NO ₂ , C (O) R ₁ , OR ₉ , SR ₉ , CN, and NR ₁₀ R ₁₁ Independently selected from;

R ₆ is H, C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, cycloalkylalkyl (eg cyclopropylmethyl), aryl, heteroaryl, arylalkyl ( For example benzyl, 2-phenylethenyl), heteroarylalkyl (eg pyridylmethyl), OR ₁₂ and NR ₁₃ R ₁₄ ;

R ₇ is selected from OR ₁₅ , SR ₁₅ , S (O) R ₁₆ , SO ₂ R ₁₇ , NR ₁₃ R ₁₄ and NR ₁₂ SO ₂ R ₆ ;

R ₈ is H, OR ₁₅ , NR ₁₃ R ₁₄ , C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, aryl, heteroaryl, arylalkyl (eg benzyl ) And heteroarylalkyl (eg pyridylmethyl);

R ₉ is selected from C ₁ -C ₄ alkyl, for example CH ₃ and CF ₃ , C (O) -alkyl, for example C (O) CH ₃ , and C (O) CF ₃ ;

R ₁₀ and R ₁₁ are the same or different and are independently selected from H, C ₁ -C ₄ alkyl and —C (O) -alkyl;

R ₁₂ is H, C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, C ₄ -C ₉ heterocycloalkylalkyl, aryl, mixed aryl and non-aryl polycycles , Heteroaryl, arylalkyl (eg benzyl) and heteroarylalkyl (eg pyridylmethyl);

R ₁₃ and R ₁₄ are the same or different and are H, C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, aryl, heteroaryl, arylalkyl (eg benzyl ), Heteroarylalkyl (eg, pyridylmethyl), amino acyl, or

R ₁₃ and R ₁₄ together with the nitrogen to which they are attached form a non-aryl polyheterocycle with C ₄ -C ₉ heterocycloalkyl, heteroaryl, polyheteroaryl, non-aromatic polyheterocycle or mixed aryl;

R ₁₅ is selected from H, C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and (CH ₂ ) _m ZR ₁₂ Become;

R ₁₆ is C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, aryl, heteroaryl, polyheteroaryl, arylalkyl, heteroarylalkyl and (CH ₂ ) _m ZR ₁₂ Is selected from;

R ₁₇ is C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, aryl, aromatic polycycle, heteroaryl, arylalkyl, heteroarylalkyl, polyheteroaryl and NR ₁₃ R Selected from ₁₄ ;

m is an integer selected from 0 to 6;

Z is selected from O, NR ₁₃ , S and S (O).

Suitably, "unsubstituted" means that no substituent is present or the only substituent is hydrogen.

Halo substituents are selected from fluoro, chloro, bromo and iodo, preferably fluoro or chloro.

Unless stated otherwise, alkyl substituents include straight and branched C ₁ -C ₆ alkyl. Examples of suitable straight and branched C ₁ -C ₆ alkyl substituents are methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, t-butyl and the like. Unless stated otherwise, alkyl substituents include unsubstituted alkyl groups and one or more suitable substituents (including acyl, cycloalkyl, halo, oxyalkyl, alkylamino, including unsaturated (ie, one or more double or triple CC bonds are present), Aminoalkyl, acylamino, and both alkyl groups substituted by OR ₁₅ , such as alkoxy). Preferred substituents for the alkyl group are halo, hydroxy, alkoxy, oxyalkyl, alkylamino and aminoalkyl.

Unless otherwise specified, cycloalkyl substituents include C ₃ -C ₉ cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. Unless stated otherwise, cycloalkyl substituents include unsubstituted cycloalkyl groups and one or more, including C ₁ -C ₆ alkyl, halo, hydroxy, aminoalkyl, oxyalkyl, alkylamino, and OR ₁₅ (eg, alkoxy) Both cycloalkyl groups substituted by suitable substituents. Preferred substituents for cycloalkyl groups are halo, hydroxy, alkoxy, oxyalkyl, alkylamino and aminoalkyl.

The above discussion of alkyl and cycloalkyl substituents also applies to the alkyl portion of other substituents, including alkoxy, alkyl amines, alkyl ketones, arylalkyl, heteroarylalkyl, alkylsulfonyl and alkyl ester substituents and the like.

Heterocycloalkyl substituents include 3- to 9-membered aliphatic rings containing 1 to 3 heteroatoms selected from nitrogen, sulfur, oxygen, such as 4- to 7-membered aliphatic rings. Examples of suitable heterocycloalkyl substituents include pyrrolidyl, tetrahydrofuryl, tetrahydrothiofuranyl, piperidyl, piperazil, tetrahydropyranyl, morpholino, 1,3-diazapan, 1,4-dia Keyboard, 1,4-oxazapan and 1,4-oxathiapan. Unless stated otherwise, the rings are C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, aryl, heteroaryl, arylalkyl (eg benzyl), heteroarylalkyl (eg pyridylmethyl) on carbon atoms , Unsubstituted or substituted by one or more suitable substituents, including halo, amino, alkyl amino, and OR ₁₅ (eg, alkoxy). Unless stated otherwise, nitrogen heteroatoms are H, C ₁ -C ₄ alkyl, arylalkyl (eg benzyl), heteroarylalkyl (eg pyridylmethyl), acyl, aminoacyl, alkylsulfonyl and arylsulfonyl Substituted or unsubstituted by

Cycloalkylalkyl substituents include compounds of the _formula- (CH ₂ ) n ₅ -cycloalkyl, where n 5 is a number from 1 to 6. Suitable alkylcycloalkyl substituents include cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl and the like. Such substituents are unsubstituted or substituted in the alkyl moiety or the cycloalkyl moiety by suitable substituents including those listed above for alkyl and cycloalkyl.

Aryl substituents include unsubstituted phenyl and C ₁ -C ₆ alkyl, cycloalkylalkyl (eg cyclopropylmethyl), O (CO) alkyl, oxyalkyl, halo, nitro, amino, alkylamino, aminoalkyl, alkyl Phenyl substituted by one or more suitable substituents including ketones, nitriles, carboxyalkyl, alkylsulfonyl, aminosulfonyl, arylsulfonyl and OR ₁₅ (eg alkoxy). Preferred substituents include C ₁ -C ₆ alkyl, cycloalkyl (eg cyclopropylmethyl), alkoxy, oxyalkyl, halo, nitro, amino, alkylamino, aminoalkyl, alkyl ketone, nitrile, carboxyalkyl, alkylsulfonyl, Arylsulfonyl and aminosulfonyl. Examples of suitable aryl groups include C ₁ -C ₄ alkylphenyl, C ₁ -C ₄ alkoxyphenyl, trifluoromethylphenyl, methoxyphenyl, hydroxyethylphenyl, dimethylaminophenyl, aminopropylphenyl, carbethoxyphenyl, Methanesulfonylphenyl and tolylsulfonylphenyl.

Aromatic polycyclos include naphthyl, and C ₁ -C ₆ alkyl, alkylcycloalkyl (eg cyclopropylmethyl), oxyalkyl, halo, nitro, amino, alkylamino, aminoalkyl, alkyl ketones, nitrile, carboxyalkyl, Naphthyl substituted by one or more suitable substituents including alkylsulfonyl, arylsulfonyl, aminosulfonyl and OR ₁₅ (eg alkoxy).

Heteroaryl substituents include compounds having 5- to 7-membered aromatic rings containing one or more heteroatoms (eg, 1 to 4 heteroatoms) selected from N, O and S. Typical heteroaryl substituents include furyl, thienyl, pyrrole, pyrazole, triazole, thiazole, oxazole, pyridine, pyrimidine, isoxazolyl, pyrazine and the like. Unless stated otherwise, heteroaryl substituents are unsubstituted or substituted on the carbon atom by one or more suitable substituents including alkyl, said alkyl substituents, and another heteroaryl substituent. The nitrogen atom is unsubstituted or substituted, for example by R ₁₃ , and particularly useful N substituents are H, C ₁ -C ₄ alkyl, acyl, aminoacyl and sulfonyl.

Aryl alkyl substituent has the formula - (CH _{2) n5} - _{aryl, - (CH 2) n5 -1} - (CH- arylalkyl) - (CH _{2) n5} - aryl or _{- (CH 2) n5 -1 -CH} ( aryl (Aryl) wherein aryl and n5 are as defined above. Examples of the arylalkyl substituents include benzyl, 2-phenylethyl, 1-phenylethyl, tolyl-3-propyl, 2-phenylpropyl, diphenylmethyl, 2-diphenylethyl, 5,5-dimethyl-3-phenylpentyl, and the like. There is this. The arylalkyl substituent is unsubstituted or substituted in the alkyl moiety or the aryl moiety, or both, as described above for the alkyl and aryl substituents.

Heteroarylalkyl substituents include groups of the _formula- (CH ₂ ) n ₅ -heteroaryl, wherein heteroaryl and n 5 are as defined above and the bridging group is linked to the carbon or nitrogen of the heteroaryl moiety, for example 2-, 3- or 4-pyridylmethyl, imidazolylmethyl, quinolylethyl, and pyrrolylbutyl. Heteroaryl substituents are unsubstituted or substituted as discussed above for heteroaryl and alkyl substituents.

Amino acyl substituents include the formulas -C (O)-(CH ₂ ) _n -C (H) (NR ₁₃ R ₁₄ )-(CH ₂ ) _n -R ₅ , wherein n, R ₁₃ , R ₁₄ and R ₅ Is as described above). Suitable aminoacyl substituents include natural and non-natural amino acids such as glycinyl, D-tryptophanyl, L-ricinyl, D- or L-homoselinyl, 4-aminobutyric acid acyl, ± -3- Amine-4-hexenoyl.

Non-aromatic polycycle substituents include bicyclic and tricyclic fused ring systems in which each ring may be 4- to 9-membered and may contain zero or one or more double and / or triple bonds. . Suitable examples of non-aromatic polycycles are decalin, octahydroindene, perhydrobenzocycloheptene, perhydrobenzo- [f] -azulene. The substituents are unsubstituted or substituted as described above for cycloalkyl groups.

Mixed aryl and non-aryl polycycle substituents include bicyclic and tricyclic fused ring systems in which each ring may be 4- to 9-membered and at least one ring is aromatic. Suitable examples of mixed aryl and non-aryl polycycles include methylenedioxyphenyl, bis-methylenedioxyphenyl, 1,2,3,4-tetrahydronaphthalene, dibenzosuberan, dihydroanthracene, 9H-fluorene There is this. The substituents are unsubstituted or substituted by nitro or as described above for cycloalkyl groups.

As polyheteroaryl substituents, each ring may be independently 5- or 6-membered, and at least one heteroatom (eg, 1, 2, 3 or 4 heteroatoms selected from O, N or S such that the fused ring system is aromatic) And fused ring systems of bicyclic and tricyclic, which may contain. Suitable examples of polyheteroaryl ring systems include quinoline, isoquinoline, pyridopyrazine, pyrrolopyridine, furopyridine, indole, benzofuran, benzothiofuran, benzindole, benzoxazole, pyrroloquinoline and the like. Unless stated otherwise, polyheteroaryl substituents include one or more alkyl on carbon atoms, one or more alkyl substituents, and substituents of the formula -O- (CH ₂ CH = CH (CH ₃ ) (CH ₂ )) _1-3 H Unsubstituted or substituted by a suitable substituent. The nitrogen atom is unsubstituted or substituted, for example by R ₁₃ , and particularly useful N substituents are H, C ₁ -C ₄ alkyl, acyl, aminoacyl and sulfonyl.

As non-aromatic polyheterocyclic substituents, each ring may be 4- to 9-membered and contains one or more heteroatoms (eg, 1, 2, 3 or 4 heteroatoms) selected from O, N or S And bicyclic and tricyclic fused ring systems that may contain 0 or one or more CC double or triple bonds. Suitable examples of non-aromatic polyheterocycles include hexitol, cis-perhydro-cyclohepta [b] pyridinyl, decahydro-benzo [f] [1,4] oxazepineyl, 2,8-dioxabicyclo [3.3.0] octane, hexahydro-thieno [3,2-b] thiophene, perhydropyrrolo [3,2-b] pyrrole, perhydronaphthyridine, perhydro-1H-dicyclopenta [b , e] piran. Unless stated otherwise, non-aromatic polyheterocyclic substituents are unsubstituted or substituted by one or more substituents including alkyl and the above alkyl substituents on a carbon atom. The nitrogen atom is unsubstituted or substituted, for example by R ₁₃ , and particularly useful N substituents are H, C ₁ -C ₄ alkyl, acyl, aminoacyl and sulfonyl.

As mixed aryl and non-aryl polyheterocycle substituents, each ring may be 4- to 9-membered, contain one or more heteroatoms selected from O, N, or S, and one or more rings must be aromatic Bicyclic and tricyclic fused ring systems are included. Suitable examples of mixed aryl and non-aryl polyheterocycles include 2,3-dihydroindole, 1,2,3,4-tetrahydroquinoline, 5,11-dihydro-10H-dibenz [b, e] [1,4] diazepine, 5H-dibenzo [b, e] [1,4] diazepine, 1,2-dihydropyrrolo [3,4-b] [1,5] benzodiazepine, 1,5 -Dihydro-pyrido [2,3-b] [1,4] diazepin-4-one, 1,2,3,4,6,11-hexahydro-benzo [b] pyrido [2,3 -e] [1,4] diazepin-5-ones. Unless stated otherwise, mixed aryl and non-aryl polyheterocyclic substituents are unsubstituted or substituted on the carbon atom by one or more suitable substituents including -N-OH, = N-OH, alkyl, and the above alkyl substituents. Do not. Nitrogen atoms are unsubstituted or substituted, for example by R ₁₃ , and particularly useful N substituents are H, C ₁ -C ₄ alkyl, acyl, aminoacyl and sulfonyl.

Amino substituents include primary, secondary and tertiary amines, and quaternary amines in salt form. Examples of amino substituents include mono- and di-alkylamino, mono- and di-aryl amino, mono- and di-arylalkyl amino, aryl-arylalkylamino, alkyl-arylamino, alkyl-arylalkylamino and the like.

Sulfonyl substituents include alkylsulfonyl and arylsulfonyl, for example methane sulfonyl, benzene sulfonyl, tosyl and the like.

Acyl substituents include the formulas -C (O) -W, -OC (O) -W, -C (O) -OW or -C (O) NR ₁₃ R ₁₄ , wherein W is R ₁₆ , H or cycloalkyl Alkyl).

Acylamino substituents include the formulas -N (R ₁₂ ) C (O) -W, -N (R ₁₂ ) C (O) -OW and -N (R ₁₂ ) C (O) -NHOH (R ₁₂ and W Substituents as defined above).

HON-C (O) -CH = C (R ₁ ) -aryl-alkyl-, which is a R ₂ substituent, is a group of the formula:

For each substituent,

R ₁ is H, halo or straight chain C ₁ -C ₄ alkyl;

R ₂ is H, C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, alkylcyclo alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl,-(CH ₂ ) _n C (O) R ₆ , amino acyl and — (CH ₂ ) _n R ₇ ;

R ₃ and R ₄ are the same or different and are independently selected from H and C ₁ -C ₆ alkyl, or

R ₃ and R ₄ together with the carbon to which they are attached represent C═O, C═S or C═NR ₈ ;

R ₅ is H, C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, aromatic polycycle, non-aromatic polycycle , Mixed aryl and non-aryl polycycles, polyheteroaryls, non-aromatic polyheterocycles, and mixed aryl and non-aryl polyheterocycles;

n, n ₁ , n ₂ and n ₃ are the same or different and are independently selected from 0 to 6 and when n ₁ is 1 to 6 each carbon atom is independently substituted with R ₃ and / or R ₄ Is not substituted or substituted;

X and Y are the same or different and are independently selected from H, halo, C ₁ -C ₄ alkyl, CF ₃ , NO ₂ , C (O) R ₁ , OR ₉ , SR ₉ , CN and NR ₁₀ R ₁₁ ;

R ₆ is H, C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, alkylcycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, OR ₁₂ and NR ₁₃ Selected from R ₁₄ ;

R ₇ is selected from OR ₁₅ , SR ₁₅ , S (O) R ₁₆ , SO ₂ R ₁₇ , NR ₁₃ R ₁₄ and NR ₁₂ SO ₂ R ₆ ;

R ₈ is selected from H, OR ₁₅ , NR ₁₃ R ₁₄ , C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, aryl, heteroaryl, arylalkyl and heteroarylalkyl Become;

R ₉ is selected from C ₁ -C ₄ alkyl and C (O) -alkyl;

R ₁₀ and R ₁₁ are the same or different and are independently selected from H, C ₁ -C ₄ alkyl and —C (O) -alkyl;

R ₁₂ is selected from H, C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, aryl, heteroaryl, arylalkyl and heteroarylalkyl;

R ₁₃ and R ₁₄ are the same or different and are H, C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and amino Independently selected from acyl;

R ₁₅ is selected from H, C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and (CH ₂ ) _m ZR ₁₂ Become;

R ₁₆ is selected from C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and (CH ₂ ) _m ZR ₁₂ ;

R ₁₇ is selected from C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and NR ₁₃ R ₁₄ ;

m is an integer selected from 0-6;

Preference is given to compounds in which Z is selected from O, NR ₁₃ , S, S (O) or a pharmaceutically acceptable salt thereof.

Useful compounds of formula (I) include those in which one of n ₂ and n ₃ is 0 and the other is 1, in particular R ₁ , X, Y, R, including R ₂ being H or —CH ₂ —CH ₂ —OH Included are compounds wherein ₃ and R ₄ are each H.

One suitable class of hydroxamate compounds is a compound of Formula IIIa or a pharmaceutically acceptable salt thereof.

Where

n ₄ is 0 to 3;

R ₂ is H, C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, cycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl,-(CH ₂ ) _n C (O) R ₆ , amino acyl and — (CH ₂ ) _n R ₇ ;

R ₅ ′ is heteroaryl, heteroarylalkyl (eg, pyridylmethyl), aromatic polycycle, non-aromatic polycycle, mixed aryl and non-aryl polycycle, polyheteroaryl, or mixed aryl and non-aryl Polyheterocycle.

Another suitable class of hydroxyxamate compounds is the compound of formula IIIa or a pharmaceutically acceptable salt thereof.

<Formula IIIa>

n ₄ is 0 to 3;

R ₂ is H, C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, cycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl,-(CH ₂ ) _n C (O) R ₆ , amino acyl and — (CH ₂ ) _n R ₇ ;

R ₅ ′ is aryl, arylalkyl, aromatic polycycles, non-aromatic polycycles and mixed aryl and non-aryl polycycles, in particular aryl, for example p-fluorophenyl, p-chlorophenyl, pOC ₁ -C ₄ alkylphenyls such as p-methoxyphenyl, and pC ₁ -C ₄ alkylphenyls, and arylalkyls such as benzyl, ortho-, meta- or para-fluorobenzyl, ortho-, meta- or para -Chlorobenzyl, ortho-, meta- or para-mono-, di- or tri-OC ₁ -C ₄ alkylbenzyl, such as ortho-, meta- or para-methoxybenzyl, m, p-diethoxybenzyl, o , m, p-trimethoxybenzyl, and ortho-, meta- or para-mono-, di- or tri-C ₁ -C ₄ alkylphenyls such as p-methyl, m, m-diethylphenyl.

Another class of interest is a compound of Formula IIIb or a pharmaceutically acceptable salt thereof.

Where

R ₂ ′ is H, C ₁ -C ₆ alkyl, C ₄ -C ₆ cycloalkyl, cycloalkylalkyl (eg cyclopropylmethyl), (CH ₂ ) _2-4 OR ₂₁ , wherein R ₂₁ is H, methyl , Ethyl, propyl and i-propyl;

R ₅ ″ is unsubstituted 1H-indol-3-yl, benzofuran-3-yl or quinolin-3-yl, or substituted 1H-indol-3-yl, for example 5-fluoro-1H-indole -3-yl or 5-methoxy-1 H-indol-3-yl, benzofuran-3-yl or quinolin-3-yl.

Another class of hydroxyxamate compounds of interest is the compound of formula IIIc or a pharmaceutically acceptable salt thereof.

Where

The ring containing Z ₁ is aromatic or non-aromatic, said non-aromatic ring being saturated or unsaturated;

Z ₁ is O, S or NR ₂₀ ;

R ₁₈ is H, halo, C ₁ -C ₆ alkyl (methyl, ethyl, t-butyl), C ₃ -C ₇ cycloalkyl, aryl (eg unsubstituted phenyl, or 4-OCH ₃ or 4-CF ₃ Phenyl substituted by, or heteroaryl (eg, 2-furanyl, 2-thiophenyl, or 2-, 3- or 4-pyridyl);

R ₂₀ is H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl-C ₃ -C ₉ cycloalkyl (eg cyclopropylmethyl), aryl, heteroaryl, arylalkyl (eg benzyl), heteroarylalkyl (Eg, pyridylmethyl), acyl (eg, acetyl, propionyl, benzoyl) or sulfonyl (eg, methanesulfonyl, ethanesulfonyl, benzenesulfonyl, toluenesulfonyl);

A ₁ is independently 1, 2 or 3 substituents that are H, C ₁ -C ₆ alkyl, —OR ₁₉ , halo, alkylamino, aminoalkyl, halo or heteroarylalkyl (eg, pyridylmethyl);

R ₁₉ is H, C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, aryl, heteroaryl, arylalkyl (eg benzyl), heteroarylalkyl (eg pyri Dimethyl) and — (CH ₂ CH═CH (CH ₃ ) (CH ₂ )) _1-3 H;

R ₂ is H, C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, cycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl,-(CH ₂ ) _n C (O) R ₆ , amino acyl and — (CH ₂ ) _n R ₇ ;

v is 0, 1 or 2;

p is 0 to 3;

q is 1 to 5 and r is 0; or

q is 0 and r is 1 to 5;

Other variable substituents are as defined above.

Particularly useful compounds of formula IIIc are compounds in which R ₂ is H or — (CH ₂ ) _p CH ₂ OH where p is 1 to 3, in particular compounds in which R ₁ is H, for example R ₁ is H And X and Y are each H, q is 1 to 3 and r is 0, or a compound wherein q is 0 and r is 1 to 3, in particular Z ₁ is NR ₂₀ . Among these compounds, R ₂ is preferably H or —CH ₂ —CH ₂ —OH, and it is preferable that the sum of q and r is 1.

Another class of hydroxyxamate compounds of interest is the compound of formula IIId or a pharmaceutically acceptable salt thereof.

Where

Z ₁ is O, S or NR ₂₀ ;

R ₁₈ is H, halo, C ₁ -C ₆ alkyl (methyl, ethyl, t-butyl), C ₃ -C ₇ cycloalkyl, aryl (eg unsubstituted phenyl, or 4-OCH ₃ or 4-CF ₃ Phenyl substituted by) or heteroaryl;

R ₂₀ is H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl-C ₃ -C ₉ cycloalkyl (eg cyclopropylmethyl), aryl, heteroaryl, arylalkyl (eg benzyl), heteroarylalkyl (Eg, pyridylmethyl), acyl (eg, acetyl, propionyl and benzoyl) or sulfonyl (eg, methanesulfonyl, ethanesulfonyl, benzenesulfonyl, toluenesulfonyl);

A ₁ is independently 1, 2 or 3 substituents that are H, C ₁ -C ₆ alkyl, —OR ₁₉ or halo;

R ₁₉ is H, C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, aryl, heteroaryl, arylalkyl (eg benzyl) and heteroarylalkyl (eg pyridyl Methyl);

p is 0 to 3;

q is 1 to 5 and r is 0; or

q is 0 and r is 1 to 5;

Other variable substituents are as defined above.

Particularly useful compounds of formula IIId are compounds in which R ₂ is H or — (CH ₂ ) _p CH ₂ OH where p is 1 to 3, in particular compounds in which R ₁ is H, for example R ₁ is H And X and Y are each H, q is 1 to 3 and r is 0, or q is 0 and r is 1 to 3. Among these compounds, R ₂ is preferably H or —CH ₂ —CH ₂ —OH, and it is preferable that the sum of q and r is 1.

In addition, the present invention relates to a compound of formula IIIe or a pharmaceutically acceptable salt thereof.

Wherein the variable substituents are as defined above.

Particularly useful compounds of formula IIIe are those wherein R ₁₈ is H, fluoro, chloro, bromo, C ₁ -C ₄ alkyl group, substituted C ₁ -C ₄ alkyl group, C ₃ -C ₇ cycloalkyl group, unsubstituted phenyl, para -A substituted phenyl or heteroaryl (eg pyridyl) ring at the position.

Another group of compounds of formula IIIe that are useful are compounds in which R ₂ is H or — (CH ₂ ) _p CH ₂ OH where p is 1 to 3, in particular compounds in which R ₁ is H, for example R ₁ Is H, X and Y are each H, q is 1-3 and r is 0, or q is 0 and r is 1-3. Among these compounds, R ₂ is preferably H or —CH ₂ —CH ₂ —OH, and it is preferable that the sum of q and r is 1. Among these compounds, p is preferably 1, and R ₃ and R ₄ are preferably H.

Another group of compounds of formula IIIe that are useful are those wherein R ₁₈ is H, methyl, ethyl, t-butyl, trifluoromethyl, cyclohexyl, phenyl, 4-methoxyphenyl, 4-trifluoromethylphenyl, 2-furanyl , 2-thiophenyl, or 2-, 3- or 4-pyridyl, wherein 2-furanyl, 2-thiophenyl, and 2-, 3- or 4-pyridyl substituents are described above for heteroaryl rings As substituted or unsubstituted) and R ₂ is H or — (CH ₂ ) _p CH ₂ OH where p is 1 to 3, in particular R ₁ is H and X and Y are each H And q is 1 to 3 and r is 0, or q is 0 and r is 1 to 3. Among these compounds, R ₂ is preferably H or —CH ₂ —CH ₂ —OH, and it is preferable that the sum of q and r is 1.

Compounds of formula IIIe wherein R ₂₀ is H or C ₁ -C ₆ alkyl (in particular H) are important members of each of the subclasses of compounds of formula Ie described above.

N-hydroxy-3- [4-[[(2-hydroxyethyl) [2- (1H-indol-3-yl) ethyl] -amino] methyl] phenyl] -2E-2-propenamide, N -Hydroxy-3- [4-[[[2- (1H-indol-3-yl) ethyl] -amino] methyl] phenyl] -2E-2-propenamide and N-hydroxy-3- [4 -[[[2- (2-methyl-1H-indol-3-yl) -ethyl] -amino] methyl] phenyl] -2E-2-propenamide or a pharmaceutically acceptable salt thereof is important to be.

In addition, the present invention relates to a compound of formula IIIf or a pharmaceutically acceptable salt thereof.

Wherein the variable substituents are as defined above.

Useful compounds of formula IIIf are compounds in which R ₂ is H or — (CH ₂ ) _p CH ₂ OH, wherein p is 1 to 3, in particular compounds in which R ₁ is H, for example R ₁ is H, X and Y are each H, q is 1-3 and r is 0, or q is 0 and r is 1-3. Among these compounds, R ₂ is preferably H or —CH ₂ —CH ₂ —OH, and it is preferable that the sum of q and r is 1.

N-hydroxy-3- [4-[[[2- (benzofur-3-yl) -ethyl] -amino] methyl] phenyl] -2E-2-propenamide or a pharmaceutically acceptable salt thereof is important Compound of Formula IIIf.

The compounds described above are generally used in the form of pharmaceutically acceptable salts. Pharmaceutically acceptable salts include, where appropriate, pharmaceutically acceptable base addition salts and acid addition salts, such as metal salts such as alkali metal and alkaline earth metal salts, ammonium salts, organic amine addition salts, and amino acid addition salts and sulfo Nate salts are included. Acid addition salts include inorganic acid addition salts (such as hydrochloride, sulfates and phosphates) and organic acid addition salts (such as alkyl sulfonates, arylsulfonates, acetates, maleates, fumarates, tartrates, citrate and lactates). ) Is included. Examples of metal salts are alkali metal salts (eg lithium salts, sodium salts and potassium salts), alkaline earth metal salts (eg magnesium salts and calcium salts), aluminum salts and zinc salts. Examples of ammonium salts are ammonium salts and tetramethylammonium salts. Examples of organic amine addition salts are salts with morpholine and piperidine. Examples of amino acid addition salts are salts with glycine, phenylalanine, glutamic acid and lysine. Sulfonate salts include mesylate, tosylate and benzene sulfonic acid salts.

Additional HDAI compounds within the scope of formula I and methods for their synthesis are disclosed in WO 02/22577, published March 21, 2002, which is incorporated herein in its entirety. Two preferred compounds within the range of WO 02/22577 are N-hydroxy-3- [4-[(2-hydroxyethyl) {2- (1H-indol-3-yl) ethyl] -amino] Methyl] phenyl] -2E-2-propenamide or a pharmaceutically acceptable salt thereof (hereinafter referred to as "compound (IV)") and N-hydroxy-3- [4-[[[2] -(2-methyl-1H-indol-3-yl) -ethyl] -amino] methyl] phenyl] -2E-2-propenamide or a pharmaceutically acceptable salt thereof (hereinafter referred to as "compound (V)") to be.

In other embodiments of the present invention, the HDAC inhibitor compounds include trapoxin and other tetrapeptides, such as chlamydocin and HC toxin; Trichostatin and its analogs (eg, trichostatin A); Apicidin; Subveroylanilide hydroxamic acid (SAHA); Oxamflatin; MS-275; Pyroxamide; Valproic acid; [4- (2-amino-phenylcarbamoyl) -benzyl] -carbamic acid pyridin-3-ylmethyl ester and derivatives thereof; Depsipeptide FR901228; CI 994; Phenylbutyrate; Sodium butyrate; Butyric acid; J. Med. Chem. 45 (9): 1778-84 (Apr 25, 2002); 3- (4-aroyl-1H-2-pyrrolyl-N-hydroxy-propenamide; ADHA compound 8;-(- Can be selected from any compound that inhibits histone deacetylases such as Depudecin; Scriptaid; and Sirtinol.

In each case where a patent application is mentioned above, matters relating to the compound are included herein. Likewise, pharmaceutically acceptable salts, corresponding racemates, diastereomers, enantiomers, tautomers, and corresponding crystal variants (if any) of the compounds disclosed above, such as the solvates disclosed therein, Hydrates and polymorphs are included. Compounds used as active ingredients in the combinations of the invention may be prepared and administered as described in the cited documents, respectively. Also included within the scope of the invention are combinations of more than two such individual active ingredients, ie pharmaceutical combinations within the scope of the invention may include three or more active ingredients.

According to certain findings of the present invention,

1. a) pyrimidylaminobenzamide compound of formula (I); And

b) one or more HDAC inhibitors

Pharmaceutical combinations,

2. Combination of a therapeutically effective amount of a pyrimidylaminobenzamide compound of formula I with an HDAC inhibitor (eg as described above), eg simultaneously or sequentially, in a subject in need of treatment or prevention of a proliferative disease -A method of treating or preventing a proliferative disease, including administering (examples of proliferative diseases include, for example, tumors, leukemia, psoriasis, restenosis, percutaneous dermatitis and fibrosis),

3. a pharmaceutical combination as defined in 1) for use in the method defined in 2) above,

4. A pharmaceutical combination as defined in 1) for use in the manufacture of a medicament for use in the method defined in 2) above.

To provide.

The utility of the combinations of the invention in the methods specified above can be demonstrated, for example, in clinical as well as animal test methods according to the methods described below.

It has now been found that the combination of pyrimidylaminobenzamide compounds and HDAC inhibitors surprisingly possesses therapeutic properties that are particularly useful in the treatment of proliferative diseases.

In another embodiment, the invention provides a therapeutically effective amount of a combination of a pyrimidylaminobenzamide compound and an HDAC inhibitor, or a combination of pharmaceutically acceptable salts or prodrugs thereof, to a mammal in need thereof. Provided is a method for treating a proliferative disease, including administering.

Preferably, the present invention comprises administering an inhibitory amount of a combination of a pyrimidylaminobenzamide compound and an HDAC inhibitor or a combination of pharmaceutically acceptable salts thereof to a mammal in need thereof. A method of treating a mammal, particularly a human, having a sexual disorder is provided.

In the description herein, the term “treatment” refers to both prophylactic or prophylactic treatments as well as curative or disease inhibiting treatments, including treatment of patients at risk of, or suspected of having, a disease. Include. In addition, the term includes treatment for the delay of the progression of the disease.

As used herein, the term "healing" means the effectiveness in treating ongoing symptom manifestations involving proliferative diseases.

The term "prophylactic" means the prevention of the onset or recurrence of a disease involving a proliferative disease.

As used herein, the term "delay of progression" refers to a patient who is a preliminary or early stage of the disease to be treated (eg, diagnosed as a pre-form of the corresponding disease, for example during medical treatment or due to contingency symptoms). Administration of the active compound to a patient in a state where the disease is likely to progress.

This unpredictable range of properties means that the use of a combination of pyrimidylaminobenzamide compounds and HDAC inhibitors is particularly important in the manufacture of a medicament for the treatment of proliferative diseases.

In order to demonstrate that the combination of the pyrimidylaminobenzamide compound and the HDAC inhibitor is particularly suitable for the treatment of proliferative diseases with good therapeutic limits and other advantages, clinical trials can be conducted in a manner known to those skilled in the art.

A. Combination Therapy

Suitable clinical studies are, for example, open label dose escalation studies in patients with proliferative diseases. In particular, these studies demonstrate the synergy of the active ingredients of the combinations of the invention. The beneficial effect can be measured directly through the results of these studies known per se to those skilled in the art. In particular, such studies are suitable for comparing the effects of monotherapy with the active ingredients of the invention and the combination of the invention. Preferably, the dosage of formulation (a) is raised until the maximum tolerated dose is reached, and formulation (b) is administered at a fixed dosage. Alternatively, formulation (a) is administered at a fixed dosage and the dosage of formulation (b) is raised. Each patient is administered a dose of formulation (a) daily or intermittently. The efficacy of treatment in this study can be measured after 12, 18 or 24 weeks, for example by assessing symptom scores every six weeks.

Administration of the pharmaceutical combinations of the invention, for example, alleviates symptoms, delays the progression, or ameliorates the symptoms as compared to monotherapy in which only one of the pharmaceutically active ingredients used in the combinations of the invention is administered. Not only does it produce a beneficial effect (e.g., a synergistic therapeutic effect) with regard to inhibition, it also has surprising additional beneficial effects such as little side effects, improved quality of life, or reduced morbidity.

A further advantage is that lower dosages of the active ingredients of the combinations of the invention can be used (eg, dosages are generally less, administered less frequently), thereby reducing the incidence or severity of side effects. This is consistent with the purpose and needs of the patient to be treated.

As used herein, the term “co-administration” or “combined administration” and the like means to include administration of a selected therapeutic agent to a single patient and includes a therapeutic regimen that does not necessarily administer the agent in the same route of administration or at the same time. do.

One object of the present invention is to provide a pharmaceutical composition comprising the combination of the present invention in an amount that is cooperatively therapeutically effective in targeting or preventing proliferative diseases. In this composition, Formulation (a) and Formulation (b) may be administered in one combination unit dosage form or in two separate unit dosage forms, administered together, one after another, or administered separately. have. In addition, the unit dosage form may be a fixed combination.

According to the invention, a pharmaceutical composition for administering formulation (a) and formulation (b) separately or in a fixed combination (ie, a single herbal composition comprising two or more combination partners (a) and (b)) ) Can be prepared in a manner known per se, by one or more pharmacologically active combination partners alone (eg, as indicated above), or by one or more pharmaceutically acceptable carriers or diluents (especially, Suitable for enteral or parenteral administration), and suitable for enteral administration (eg, oral or rectal administration) and parenteral administration to mammals (warm-blooded animals), including humans.

Suitable pharmaceutical compositions contain, for example, from about 0.1% to about 99.9%, preferably from about 1% to about 60% of the active ingredient (s). Pharmaceutical formulations for combination treatment for enteral or parenteral administration are, for example, in unit dosage forms such as sugar-coated tablets, tablets, capsules or suppositories, or ampoules. Unless indicated otherwise, they are prepared in a manner known per se, for example by conventional mixing, granulating, sugar-coating, dissolving or lyophilizing processes. It will be appreciated that since the required effective amount can be achieved by the administration of multiple dosage units, the unit content of the combination partner contained in the individual dosages of each dosage form need not constitute itself an effective amount.

In particular, a therapeutically effective amount of each of the combination partners of the combinations of the invention may be administered simultaneously or sequentially and in any order, and the components may be administered separately or in a fixed combination. For example, a method of preventing or treating a proliferative disease according to the present invention may comprise (i) a first agent in free form or in a pharmaceutically acceptable salt form (a); And (ii) preparations in free form or in pharmaceutically acceptable salt form (b) simultaneously or sequentially in any order, in a co-therapeutically effective amount, preferably in a synergistically effective amount, in a dose corresponding to the amount described herein. For example daily or intermittent administration. Individual combination partners of the combinations of the invention may be administered separately or simultaneously at different times during the course of treatment in the form of divided or single combinations. Moreover, the term "administration" also encompasses the use of prodrugs of combination partners that are converted to the combination partner itself in vivo. Accordingly, the present invention should be construed to encompass all such concurrent or alternating treatment regimens, and the term “administration” should be interpreted accordingly.

The effective dosage of each combination partner used in the combinations of the present invention may vary depending on the particular compound or pharmaceutical composition used, the mode of administration, the disease being treated, and the severity of the disease being treated. Thus, the dosage regimen of the combination of the present invention is selected according to various factors including the mode of administration and the kidney function and liver function of the patient. The skilled clinician or physician can readily determine and prescribe the effective amount of a single active ingredient required to alleviate, ameliorate or arrest the progress of the disease. Optimum accuracy in achieving concentrations of the active ingredient within the range of gaining efficacy without toxicity requires a therapy based on the dynamics of the availability of the active ingredient to the target site.

The daily dosage for formulation (a) or (b) will of course vary depending on various factors, such as the compound selected, the particular disease being treated and the desired effect. Generally, however, formulation (a) is about 0.03 to 5 mg / kg / day, in particular 0.1 to 5 mg / kg / day (eg 0.1 to 2.5 mg / kg / day) as a single or divided dose. Satisfactory results are achieved when administered in a daily dose of. Formulations (a) and (b) may be by any conventional route, in particular, enterally, for example orally (e.g. in the form of tablets, capsules, drinking solutions) or parenterally (e.g., injection solutions). Or in the form of a suspension). Thus, suitable dosage forms for oral administration include from about 0.02 to 50 mg of active ingredient, typically from 0.1 to 30 mg, for example formulation (a) or (b) with one or more pharmaceutically acceptable diluents or carriers. Include.

Formulation (b) may be administered to a human in a daily dosage range of 0.5 to 1,000 mg. Thus, unit dosage forms suitable for oral administration comprise from about 0.1 to 500 mg active ingredient with one or more pharmaceutically acceptable diluents or carriers.

Administration of the pharmaceutical combinations of the invention, compared to monotherapy in which only one of the pharmaceutically active ingredients used in the combinations of the invention, inhibits uncontrolled proliferation of blood stem cells or chronic myelogenous leukemia (CML), acute Not only does it have a beneficial effect (eg, a synergistic therapeutic effect) in relation to retarding the progression or tumor proliferation of leukemias such as lymphocytic leukemia (ALL) or acute myeloid leukemia (AML), but with fewer side effects or This has surprisingly beneficial benefits, such as improved quality of life or lower morbidity.

It is a further advantage that the active ingredients of the combinations of the invention can be used at lower dosages (eg, dosages are generally less, administered less frequently) or can reduce the incidence or severity of side effects. This is consistent with the purpose and needs of the patient to be treated.

Combinations of pyrimidylaminobenzamide compounds and HDAC inhibitors, independently or together, may be combined with one or more pharmaceutically acceptable carriers, and optionally one or more other conventional pharmaceutical adjuvants, and include tablets, capsules, caplets, and the like. It may be administered intranasally (eg orally) in the form of or parenterally (eg intraperitoneally or intravenously) in the form of sterile injection solutions or suspensions. The intestinal and parenteral compositions can be prepared by conventional means.

Combinations of pyrimidylaminobenzamide compounds and HDAC inhibitors may be used alone or in combination with one or more pharmaceutically active compounds for pathological use. The active compounds may be combined into the same pharmaceutical formulation, or the combination partner may be administered independently or using a fixed combination that is different from the combination partner in different amounts (ie, administered simultaneously or at different times). May be combined in the form of a "kit of parts" which is a combined preparation in view. Parts of the kit of parts may be administered, for example, simultaneously or alternately in chronological order, ie at different time points at the same or different time intervals for any part of the kit of parts. Non-limiting examples of compounds that can be used in combination with pyrimidylaminobenzamide compounds and HDAC inhibitors include cytotoxic chemotherapeutic drugs such as cytosine arabinoside, daunorubicin, doxorubicin, cyclophosphamide, VP-16 Or imatinib. In addition, the combination of pyrimidylaminobenzamide compounds and HDAC inhibitors can be combined with other signal transduction inhibitors or other oncogene-targeting drugs, in which case significant synergy is expected.

B. Diseases Treated

The term "proliferative disease" includes, but is not limited to, tumors, psoriasis, restenosis, percutaneous dermatitis and fibrosis.

The term "blood malignancy" refers in particular to the expression of leukemia, in particular Bcr-Abl, c-Kit or HDAC (or dependent on Bcr-Abl, c-Kit or HDAC), CML and ALL, in particular Philadelphia Chromosome positive ALL (Ph + ALL) and imatinib-resistant leukemia. In particular, it is preferred to use the combinations of the invention in leukemias such as CML, ALL or AML. In particular, it is most preferred for use in diseases resistant to imatinib (Imatinib is marketed under the name Gleevec®).

The term “solid tumor disease” especially refers to ovarian cancer, breast cancer, colon cancer and generally gastrointestinal cancer, cervical cancer, lung cancer (eg small cell lung cancer and non-small cell lung cancer), head and neck cancer, bladder cancer, prostate cancer or Kaposi's sarcoma. sarcoma).

Thus, combinations according to the invention which inhibit the mentioned protein kinase activity, in particular the activity of the tyrosine protein kinases mentioned above and below, can be used for the treatment of protein kinase dependent diseases. Protein kinase dependent diseases are particularly proliferative diseases, preferably benign or especially malignant tumors (e.g. kidney, brain, liver, adrenal gland, bladder, breast, stomach (especially gastrointestinal tumors), ovaries, colon, rectum, prostate , Pancreas, lung (especially SCLC), carcinoma of the vagina or thyroid, sarcoma, multiple myeloma, glioblastoma and many tumors of the head and neck, and leukemia), in particular colon carcinoma or colorectal adenoma, or tumors of the head and neck, epidermal hyperplasia (Especially psoriasis), prostate hyperplasia, and especially epithelial neoplasia (eg breast carcinoma) or leukemia. They can degenerate tumors and prevent the formation of tumor metastases and the proliferation of (also fine) metastases. They can also be used for the treatment of epidermal hyperplasia (eg psoriasis), prostate hyperplasia, and especially epithelial neoplasia (eg breast carcinoma). It is also possible to use the combinations of the invention in the treatment of immune system diseases, as long as some or in particular individual tyrosine protein kinases are involved, furthermore, the combinations of the invention are particularly mentioned at least one tyrosine protein kinase, in particular Where signal transduction by selection from those involved is involved, it can be used for the treatment of central or peripheral nervous system diseases.

In CML, the mutually balanced chromosomal translocation in hematopoietic stem cells (HSC) produces the Bcr-Abl hybrid gene. The latter encodes a carcinogenic Bcr-abl fusion protein. Abl encodes a tightly regulated protein tyrosine kinase, which plays a fundamental role in regulating cell proliferation, adhesion and apoptosis, while the Bcr-Abl fusion gene encodes a constitutively activated kinase, This transforms HSCs to produce phenotypes that indicate deregulated clonal proliferation, reduced ability to attach to bone marrow substrates, and reduced apoptosis response to mutagenesis stimuli, allowing progressively more malignant transformations to accumulate. have. The resulting granulocytes do not develop into mature lymphocytes and are released into the circulation, resulting in increased susceptibility to defects and infection of mature cells. ATP-competition inhibitors of Bcr-Abl have been described, which prevents kinases from activating mitotic and anti-apoptotic pathways (eg, P-3 kinase and STAT5), leading to the death of Bcr-Abl phenotype cells Thus providing an effective treatment regimen for CML. Thus, the combinations of the present invention are particularly suitable for the treatment of diseases associated with their overexpression, in particular leukemia (eg leukemias such as CML or ALL).

In a broad aspect of the present invention, proliferative diseases include hyperproliferative diseases such as leukemia, hyperplasia, fibrosis (particularly other types of fibrosis such as pulmonary fibrosis, and also renal fibrosis), angiogenesis, psoriasis, atherosclerosis and Vascular smooth muscle proliferation (eg, stenosis or restenosis after angioplasty). In another aspect, the combinations of the present invention can be used to treat arthritis.

In addition, the combinations of the present invention treat or prevent fibrotic disorders such as scleroderma (systemic sclerosis), protein aggregation and amyloid formation, such as Huntington's disease, and inhibit the replication of hepatitis C virus It can be used to treat hepatitis C virus, to treat tumors associated with viral infections such as human papilloma virus, and to suppress viruses that are dependent on heat-shock protein.

The combination of the present invention mainly inhibits the proliferation of blood vessels, so that a number of diseases associated with unregulated angiogenesis, in particular diseases caused by ocular neovascularisation, in particular retinopathy such as diabetic retinopathy or age- Related macular degeneration, psoriasis, hemangioblastomas such as hemangiomas, hemangioblast proliferative disorders such as chronic or acute kidney disease such as diabetic nephropathy, malignant nephropathy, thrombotic microangiopathy syndrome or transplant rejection, or in particular Inflammatory kidney diseases such as glomerulonephritis, in particular mesangioproliferative glomerulonephritis, hemolytic-uremic syndrome, diabetic nephropathy, hypertensive nephropathy, atherosclerosis, arterial restenosis, autoimmune disease, diabetes, endometriosis, Chronic asthma, and especially neoplastic diseases (solid tumors, also leukemia and other hematologic malignancies), such as, in particular, breast cancer, texture Cancer, lung cancer is effective for (especially small-cell lung cancer), prostate cancer, or Kaposi's sarcoma. The combinations of the present invention inhibit the proliferation of tumors and are particularly suitable for preventing metastatic spread of tumors and proliferation of micrometastases.

Combinations of the invention in particular,

(i) breast tumors; Squamous cell tumors, such as squamous cell head and / or cervical tumors or oral tumors; Lung tumors, such as small cell or non-small cell lung tumors; Gastrointestinal tumors such as colorectal tumors; Or genitourinary tumors such as prostate tumors, in particular hormone-refractory prostate tumors;

(ii) proliferative diseases refractory to treatment with other chemotherapeutic agents; or

(iii) tumors refractory to treatment with other chemotherapeutic agents due to multidrug resistance

Can be used to treat

Reagents and Antibodies: Compounds (V) and (II) were provided by Novartis Pharmaceuticals (East Hanover, NJ). Polyclonal anti-PARP, anti-caspase-9, anti-caspase-3 and anti-p-ERK1 / 2 antibodies were purchased from Cell Signaling Technology (Beverly, Mass.). Polyclonal anti-STAT-5 and goat polyclonal anti-Pim-2 antibodies, and monoclonal anti-c-Myc and anti-Abl antibodies, were described as Santa Cruz Biotechnology (Santa Cruz, CA, USA). ). Monoclonal anti-p-STAT5 antibodies were purchased from Upstate Biotechnology, Lake Placid, NY. Antibodies for immunoblotting analysis of p21, p27, p-CrkL, CrkL, p-AKT, AKT, Bim, Bcl-x _L and ERK1 / 2 were obtained as described above.

Cell Lines and Cell Cultures: CML LAMA-84 and K562 cells expressing Bcr-Abl were obtained from American Tissue Culture Collection (Manassas, Va.) And contained 10% fetal bovine serum The cells were maintained in culture in RPMI medium and passaged twice a week. Mouse pro-B BaF3 cells were cultured in RPMI-1640 complete medium supplemented with 10% WEHI medium as a source of IL-3. For the studies described herein, cells growing exponentially were exposed to the indicated concentrations of compound (II) and / or compound (V). After this treatment, cells or cell pellets were washed without drug (s) and the study performed.

Site-directed mutagenesis and nucleofection: Three p210 Bcr-Abl constructs were used in this study. p210 Bcr-Abl WT and p210 Bcr-Abl (T315I) constructs were generated. P210 Bcr-Abl (E255K) by site-directed mutagenesis of the Bcr-Abl containing pSVneo construct using the QuikChange II XL kit (Stratagene, Cedar Creek, TX) according to the manufacturer's instructions ) Mutants were generated and the resulting clones were sequenced to identify point mutations. To nucleofect p210 Bcr-Abl constructs into BaF3 cells, cuvette 5 × 10 ⁶ BaF3 cells in 100 μL of Nucleofector Solution V (Amaxa, Gatorsburg, MD) 5 μg of p210 Bcr-Abl WT, p210 Bcr-Abl (T315I), or p210 Bcr-Abl (E255K), and nucleofection using program G-16. Following nucleofection, cells were recovered by incubating overnight at a concentration of 1 × 10 ⁶ cells / mL in RPMI-1640 complete medium supplemented with 10% WEHI medium as a source of IL-3. Stable transfectants of BaF3 cells expressing WT or mutant forms of Bcr-Abl (ie, T315I or E255K) were supplemented with 10% serum, 1.0 U / mL penicillin, 1 μg / mL streptomycin and 0.75 mg / mL G418 It was maintained in one RPMI 1640 medium. Subsequently, stably expressing cells were further selected by removing IL-3. After confirming Bcr-Abl expression by immunoblotting assay, cells were used for the studies described herein.

Primary CML-BC cells and NBMC: obtained from peripheral blood and / or bone marrow of 10 patients who met the clinical criteria of imatinib-resistant Ph chromosome-positive CML-BC (imatinib-resistant Philadelphia chromosome-positive chronic myelogenous leukemia-onset) Leukemia cells were harvested and purified. In addition, NBMCs were harvested and purified. As part of a clinical protocol approved by the University of South Florida Institutional Review Board (IRB), all patients signed a notice agreement to use the cells for the experiment.

Sequencing of Bcr-Abl in CML-BC Cells: Using the Trizol Method (Invitrogen, Carlsbad, CA), Bcr is not responding to the treatment of imatinib and Compound (II) Total RNA was isolated from 10-15 × 10 ⁶ cells available from two patients believed to have the -Abl T315I mutation. Total RNA (5 μg) was reverse transcribed with a first-strand cDNA synthesis kit (Invitrogen). Reverse-transcribed cDNA was used for amplification of the polymerase chain reaction (PCR) to amplify the fragment of Bcr-Abl containing the Bcr junction region and c-Abl kinase region. Amplified sequences were agarose gel-purified and cloned into pCR4-TOPO plasmid. The resulting plasmid was transferred to Escherichia at 37 ° C. coli ) were transformed overnight with Mach1 cells (Invitrogen). Ten colonies of each sample were identified by colony PCR and subcultured for plasmid isolation. Sequences of the isolated plasmids were identified with T3 and T7 primers for c-Abl kinase domain mutations.

Suspension culture or colony proliferation inhibition: Treatment with indicated concentrations of Compound (II) and / or Compound (V) for 48 hours, then drug-treated and untreated cells were washed in RPMI 1640 medium. The cells were then placed in suspension culture for 4 days at a concentration of 200,000 cells / mL. At the end of the incubation period, cell concentration and cell number growth rate were measured. Alternatively, about 200 cells treated with the drug and then treated under each condition are resuspended in 100 μL of RPMI 1640 medium containing 10% FBS, followed by 1.0 mL metocult per well according to the manufacturer's protocol. The wells were placed in 12-well plates containing Methocult medium (Stem Cell Technologies, Vancouver, Canada). The plate was placed in an incubator for 10 days at 37 ° C. with 5% CO ₂ . Following incubation, colonies of 50 or more cells in each well were counted by inverted microscope and the percentage of colony growth inhibition compared to untreated control cells was calculated.

% Evaluation of Non-Survival Cells: Cells were stained with trypan blue (Sigma, St. Louis, MO). The cells showing trypan blue uptake in the hemocytometer were counted to determine the number of non-viable cells and reported in% of untreated control cells.

Apoptosis Evaluation by Annexin V Staining: Drug-treated and untreated cells were stained with Annexin V and PI and% of apoptosis cells were measured by flow cytometry. Analysis of the synergy between Compound (II) and Compound (V) in inducing apoptosis of K562 and LAMA-84 cells was commercially-used by median dose-effect analysis of Chou and Talalay. Possible software (Calcusyn; Biosoft, Ferguson, Missouri).

Western analysis of proteins: Western analysis was performed using specific antiserum or monoclonal antibodies according to the protocols recorded above, and horizontal scanning density was performed on Western blots.

Immunoprecipitation and Immunoblotting Analysis of Bcr-Abl: After designated drug treatment, cells were lysed in lysis buffer (20 mM Tris [pH 8], 150 nM sodium chloride, 1% NP40, 0.1 M sodium fluoride, 1 for 1 min on ice). mM PMSF, 1 mM sodium orthovanadate, 2.5 μg / mL leupetin, 5 μg / mL aprotinin), and nuclei and cytoplasmic debris were removed by centrifugation. Cell lysates (200 μg) were incubated with Abl-specific monoclonal antibodies at 4 ° C. for 1 hour. To this, washed Protein G agarose beads were added and incubated at 4 ° C. overnight. Immunoprecipitates were washed three times in lysis buffer, proteins were eluted with SDS sample loading buffer, and immunoblotting assays were performed with specific antibodies against anti-Abl or antiphosphotyrosine antibodies.

Statistical Analysis: Significant differences between values obtained from leukemia cell populations treated with different experimental conditions were determined using the Student's t test. Significance is given when the P value is less than 0.05.

Compounds (II) and (V) induce apoptosis of K562 and LAMA-84 cells: Determine the effect of Compound (II) and / or Compound (V) on cultured primary CML-BC cells. Apoptotic effects by treatment with Compound (V) or Compound (II) alone on K562 and LAMA-84 cells were determined. Upon exposure to compound (V) or compound (II) alone, apoptosis of K562 and LAMA-84 cells was induced in a dose-dependent manner. The data also show that Compound (II) is about 10 times more potent than imatinib in inducing apoptosis of K562 and LAMA-84 cells. When LAMA-84 cells were treated with Compound (II), the levels of tyrosine phosphorylated Bcr-Abl were inhibited in a dose-dependent manner and did not affect the levels of Bcr-Abl. Treatment with Compound (II) also inhibited the level of p-CrkL (see below), suggesting that AMN107 inhibits the TK activity of Bcr-Abl. Treatment with Compound (II) attenuated the levels of p-STAT5 and decreased the expression of c-Myc and Bcl-xL transactivated by STAT5. Treatment with Compound (II) also inhibited the level of p-AKT (but not AKT) associated with induction of p27 levels. The results were also observed after exposure to imatinib. Similar effects of Compound (II) were also observed in K562 cells.

Combination of Compound (V) with Compound (II) shows excellent anti-Bcr-Abl activity and synergistically induces apoptosis of K562 and LAMA-84 cells: for Bcr-Abl and then for Bcr-Abl The effect of the combination of compound (V) and compound (II) on the level of downstream signal transduction protein was determined. Relatively low concentrations of Compound (V) (20 nM) and Compound (II) (50 nM) intensified the depletion of Bcr-Abl in K562 cells over 24 hours, compared to treatment with the formulation alone, and Many p27 levels were induced. In contrast, p21 levels were induced to a similar degree by the combined treatment of compound (II) and compound (V) compared to treatment with compound (V) alone. In addition, the combination treatment of Compound (V) and Compound (II) further weakened the levels of p-CrkL, Bcl-xL and c-Myc, but further induced Bim. Combination of compound (II) with compound (V) followed by simultaneous induction of Bim and attenuation of Bcl-xL is associated with more PARP cleavage, which is associated with effector caspases 3 and 7 in apoptosis. Due to increased activity. Similar effects of Compound (V) and Compound (II) were also observed for LAMA-84 cells. Apoptotic effects of Compound (V) and / or Compound (II) on suspension culture and colony proliferation of K562 cells were determined. Combination of Compound (II) and Compound (V) significantly inhibited colony proliferation compared to treatment with drug alone (P <0.05). Similar effects of the combinations were also observed for suspension culture proliferation of K562 cells. Apoptosis effect (% increase in Annexin V-positive cells) by the combination treatment of Compound (II) and Compound (V) on K562 and LAMA-84 cells was determined. Notably, as determined by median dose-effect isobologram analysis, exposure to a combination of Compound (II) and Compound (V) resulted in a synergistic apoptosis effect in K562 and LAMA-84 cells. . Combination index values for Compound (II) and Compound (V) were less than 1.0 for each cell type. The CI values for K562 were 0.47, 0.36, 0.45, 0.45 and 0.45, respectively, and the CI values for LAMA84 were 0.85, 0.22, 0.21 and 0.16, respectively. In addition, the effect of compound (II) and / or compound (V) on NBMC was determined. Compound (II) (1.0 μM or less) had no effect, but exposure to Compound (V) 20 and 50 nM for 48 hours resulted in 13.1% and 15.9% of NBMC (average of two samples obtained from two trials). Reduced survival was induced. Combination with Compound (II) did not significantly increase survival reduction of NBMCs due to exposure of Compound (V) 50 nM (P> 0.05).

Compound (V) depletes the level of mutant Bcr-Abl and induces apoptosis of IM-resistant BaF3 cells expressing Bcr-AblT315I or Bcr-AblE255K: unmutated Bcr-Abl or point mutant Bcr-AblE255K or Bcr- The effect of compound (V) and / or compound (II) treatment on BaF3 cells with ectopic expression of AblT315I was determined. Similar to the effects seen in K562 and LAMA824 cells with endogenous expression of Bcr-Abl, Compound (II) induced apoptosis of BaF3 / Bcr-Abl cells in a dose-dependent manner. In addition, combination of Compound (II) and Compound (V) induced significantly more apoptosis of BaF3 / Bcr-Abl cells compared to treatment with formulation alone. Apoptosis of BaF3 / Bcr-Abl cells exposed to imatinib was dose-dependently induced, but BaF3 / Bcr-AblT315I cells were resistant to imatinib to levels as high as 10 μM. In contrast, BaF3 / Bcr-AblT315I cells were as sensitive as BaF3 / Bcr-Abl cells to apoptosis induced by treatment with Compound (V) alone. Treatment with Compound (V) 50 nM for 48 hours induced apoptosis in about 30% of BaF3 / Bcr-Abl T315I cells. Lower levels of LBH589 were less effective. In contrast, BaF3 / Bcr-AblT315I cells were resistant to high levels of Compound (II) as high as 2,000 nM. Notably, combination with Compound (II) 2,000 nM (but not 100 nM) significantly increased Compound (V) -induced apoptosis of BaF3 / Bcr-AblT315I cells. For BaF3 / Bcr-AblE255K cells, 100 nM of Compound (II) was not effective, but exposure to Compound (II) 200 and 500 nM induced apoptosis of 26.0% and 43.0% of cells, respectively. In addition, combination of Compound (II) (500 nM) and Compound (V) (50 nM) induced significantly more apoptosis of BaF3 / Bcr-AblE255K cells compared to treatment with formulation alone (P <0.01). (Combination with 100 nM of compound (II) was less effective). In addition, combinations with higher concentrations of Compound (II) (1.0 or 2.0 μM) also enhanced Compound (V) -induced apoptosis of BaF 3 / Bcr-AblE255K. The apoptosis effect of compound (II) and / or compound (V) is related to their effect on Bcr-Abl levels of BaF3 / Bcr-Abl, BaF3 / Bcr-AblE255K and BaF3 / Bcr-AblT315I cells. Treatment with any level of Compound (II) tested alone did not lower the Bcr-Abl levels of any of these three cell types. In addition, exposure to compound (II) did not affect the level of p-CrkL or CrkL. In contrast, exposure to Compound (V) 50 nM for 24 hours resulted in a decrease in Bcr-Abl levels in all three BaF3 transfectants. Notably, co-treatment with Compound (V) and Compound (II) induced more depletion of Bcr-Abl in BaF3 / Bcr-Abl cells compared to treatment with formulation alone. Notably, the combination treatment of Compound (V) and Compound (II) further reduced Bcr-AblT315I and Bcr-Abl E255K levels in BaF3 / Bcr-AblT315I and BaF3 / Bcr-AblE255K cells, respectively. Similar effects were observed for p-CrkL (but not CrrkL) levels.

Combination of Compound (II) with Compound (V) weakens Bcr-Abl more and reduces the survival of primary imatinib-resistant CML cells compared to the formulation alone: patients with recurring imatinib-resistant CML-BC The anti-leukemic effect of Compound (V) and / or Compound (II) on primary CML cells isolated from 10 peripheral blood and / or bone marrow samples was determined. Three of these samples were found to express Bcr-AblT315I (samples 8, 9 and 10). Among the samples of the remaining IM-refractory primary CML cells (samples 1-7), the mutation status of Bcr-Abl could not be determined due to inadequate sample size. Both compounds (II) and compound (V) in Samples 1 to 7 induced a decrease in cell viability, which was dose dependent. In addition, in this sample, combining Compound (V) (20 or 50 nM) with Compound (II) induced a further decrease in cell viability compared to treatment with the formulation alone. Sample 7 was relatively resistant to low concentrations of compound (II) but was sensitive to compound (V). In three samples with Bcr-AblT315I mutations (Samples 8, 9 and 10), treatment with Compound (II) did not increase the decrease in cell viability, whereas cell survival when exposed to Compound (V) alone for 48 hours Figures were significantly inhibited in a dose dependent manner. Notably, in the samples (8, 9 and 10), co-treatment with Compound (II) 50 or 100 nM did not lead to a reduction in Compound (V) -induced cell viability. In one sample (No. 9), exposure to 2.0 μM of compound (II) had no effect, but compared to that induced by 42.0% apoptosis of cells treated with compound (V) 50 nM alone, Compound (V) 50 Combination of nM with 2.0 μM of compound (II) induced 63.7% apoptosis of cells. Western blotting analysis of the whole cell lysate of sample 5 showed that Bcr-Abl, p, compared with treatment with the formulation alone when combined with 50 nM of compound (V) and 100 nM of compound (II) for 24 hours -CrkL and p-STAT5 were further weakened. In contrast, in Sample 9, even treatment with 1000 nM of Compound (II) alone had little effect on the levels of Bcr-Abl, p-CrkL and p-STAT5, whereas Compound (V) 50 nM and Compound (II) When combined with, the levels of Bcr-AblT315I, p-CrkL and p-STAT5 were markedly depleted. This finding is similar to the finding in BaF3 cells with ectopic expression of Bcr-AblT315I.

In the above examples, when treated with a combination of all HDAC inhibitor compounds (V) and Bcr-Abl TK inhibitor compounds (II), cultured mouse pro with ectopic and endogenous expression of unmutated Bcr-Abl, respectively It was demonstrated that apoptosis of -B BaF3 and human CML cells was synergistically induced. In addition, the combination is more active against BaF3 cells and imatinib-resistant primary CML cells with ectopic expression of mutant Bcr-AblE255K or Bcr-AblT315I compared to drug alone.

Claims (12)

a) pyrimidylaminobenzamide compounds of formula I; And b) one or more histone deacetylase inhibitors Pharmaceutical combinations comprising. The compound of claim 1 wherein formulation a) is 4-methyl-3-[[4- (3-pyridinyl) -2-pyrimidinyl] amino] -N- [5- (4-methyl- of formula II): Pharmaceutical combinations selected from 1H-imidazol-1-yl) -3- (trifluoromethyl) phenyl] benzamide. <Formula II>

The compound of claim 2, wherein the formulation b) is N-hydroxy-3- [4-[(2-hydroxyethyl) {2- (1H-indol-3-yl) ethyl] -amino] methyl ] Phenyl] -2E-2-propenamide or a pharmaceutically acceptable salt thereof, N-hydroxy-3- [4-[[[2- (2-methyl-1H-indol-3-yl) of the formula ) -Ethyl] -amino] methyl] phenyl] -2E-2-propenamide or a pharmaceutically acceptable salt thereof, and combinations thereof. <Formula IV>

<Formula V>

The method of claim 2, wherein the formulation b) is trapoxin, tetrapeptide, chlamydocin, HC toxin, trichostatin, trichostatin A, apicidin, subveroilanilide Hydroxamic acid (SAHA), oxamflatin, MS-275, pyroxamide, valproic acid, [4- (2-amino-phenylcarbamoyl) -benzyl] -carbamic acid pyridine- 3-ylmethyl ester and derivatives thereof, Depsipeptide, FR901228, CI-994, phenylbutyrate, sodium butyrate, butyric acid, 3- (4-aroyl-1H-2-pyrrolyl-N-hydroxy-pro Pharmaceutical combinations selected from phenamide, ADHA compound 8,-(-) depudecin, Scriptaid and Sirtinol. Use of a pharmaceutical combination according to claim 1 for the manufacture of a medicament for the treatment or prevention of proliferative diseases. 6. Use according to claim 5, wherein the disease is leukemia. Use according to claim 5, wherein the disease is chronic myeloid leukemia or acute lymphocytic leukemia. Subjects in need of treatment or prevention of a proliferative disease, comprising co-administering a therapeutically effective amount of at least one histone deacetylase inhibitor with a pyrimidylaminobenzamide compound of formula (I), for example simultaneously or sequentially A method of treating or preventing a proliferative disease. The method of claim 8, wherein the disease is leukemia. Histone deacetylase inhibitors and 4-methyl-3-[[4- (3-pyridinyl) -2-pyrimidinyl] amino] -N- [5- (4-methyl-1H-imidazol-1-yl A method of treating leukemia, comprising administering a combination of) -3- (trifluoromethyl) phenyl] benzamide. N-hydroxy-3- [4-[(2-hydroxyethyl) {2- (1H-indol-3-yl) ethyl] -amino] methyl] phenyl] -2E-2-propene Amide or a pharmaceutically acceptable salt thereof, N-hydroxy-3- [4-[[[2- (2-methyl-1H-indol-3-yl) -ethyl] -amino] methyl] phenyl ] -2E-2-propeneamide or a pharmaceutically acceptable salt thereof, and leukemia comprising administering a combination of a histone deacetylase inhibitor selected from a combination thereof and a pyrimidylaminobenzamide compound of formula (I) How to treat it. <Formula IV>

<Formula V>

The method according to claim 10, wherein the histone deacetylase preparation is used for trapoxine and other tetrapeptides such as chlamycin and HC toxin, trichostatin and analogs thereof, such as trichostatin A, apicidine, subveroilanilide hydroxy Triacid (SAHA), oxamplatin, MS-275, pyroxamide, valproic acid, [4- (2-amino-phenylcarbamoyl) -benzyl] -carbamic acid pyridin-3-ylmethyl ester and derivatives thereof , Depsipeptide FR901228, CI-994, phenylbutyrate, sodium butyrate, butyric acid, 3- (4-aroyl-1H-2-pyrrolyl-N-hydroxy-propenamide, ADHA compound 8,-(-) depu The process selected from descin, scriptide and sirtinol.